The present invention relates generally to detecting and isolating target molecules and, more specifically, to detecting and isolating phosphomolecules, nitrotyrosine-containing molecules and sulfated molecules.
Cells of the body contain many types of molecules that vary in function, size, lifetime, and numerous other characteristics. Some of these molecules are unchanged during their lifetime within the body, while other molecules become modified through chemical reactions. The modifications can be indicative of particular cell states, including normal states as well as abnormal states caused by injury, infection and disease.
In particular, many proteins are chemically modified during their lifetime in the body. Accordingly, a protein can include any number of modifications that occur during and after its synthesis within the cell. These modifications change the size and the structure of the protein, thereby often changing the protein's function or behavior in the cell. An example of a possible modification of a protein is enzymatic cleavage of the original polypeptide by proteases to produce a smaller product. Other modifications include the addition of sugar molecules to certain amino acids in the protein (glycosylation), the addition of a phosphate group (phosphorylation), the addition of a sulfate group (sulfation) and the addition of a nitro group on a tyrosine residue (tyrosine nitrosylation).
Reversible phosphorylation of threonine, serine, and tyrosine residues by enzymes called kinases (which add a phosphate) and phosphatases (which remove the phosphate) plays an important role in regulating many cell processes, such as growth and cell cycle control. Phosphorylation can occur sequentially from one protein to another, resulting in a series of activations called a “phosphorylation cascade.” Phosphorylation cascades are recognized as signaling networks that direct growth, death, and differentiation of cells—the critical signals for maintaining normal cells in the body. Efforts are underway within the research community to identify phosphorylated proteins of various cell types, the population of phosphorylated proteins being referred to as the “phosphoproteome.” Increased understanding of protein phosphorylation has already led to development of effective cancer therapies, such as Herceptin® and Erbitux®.
Nitrotyrosine has been shown to be present in proteins from a variety of clinical conditions including atherosclerotic lesions of human coronary arteries, postischemic heart, and placenta during preeclampsia, inflammatory disease, and neurological disease, such as amyotrophic lateral sclerosis (ALS). Researchers also have reported that nitrotyrosine levels were reduced among patients treated with statins, a commonly used class of cholesterol-lowering drugs, indicating that nitrotyrosine serves as a good measure for monitoring the anti-inflammatory effects of statins. For these reasons, among many others, it is of interest to the scientific and medical communities to better understand the role of tyrosine nitrosylation in normal and disease conditions of the body.
Sulfation has a well-established role in drug metabolism and chemical defense. In this regard, many chemicals to which we are exposed are rendered more biologically active following metabolism, and this bioactivation is central to the mechanism of action of numerous cancer-causing agents, including those in the diet. Sulfation is the terminal step in the bioactivation of numerous cancer-causing agents. To understand abnormal sulfation in the body, researchers are currently studying human genes for sulfation enzymes. As an example, studies have focused on the possibility that certain sulfation enzyme gene mutations can serve as risk factors in cancer of the bladder and colon, which represent target tissues for cancer-causing agents that are activated by sulfation. As an example of normal sulfation in the body, certain hormones such as catecholamines circulate predominantly as their sulfate conjugates (more than 95% of circulating dopamine is in the form of dopamine sulfate). An increased understanding of sulfation that occurs in normal and disease conditions is expected to provide further insight into drug metabolism and chemical defense, as well as into normal and disease conditions of the body.
Thus, detecting and isolating modified forms of molecules, such as proteins, is of interest to those doing research to understand normal and abnormal conditions of cells and systems of the body, as well as to those developing tests for diagnosis and prognosis of abnormal conditions relating to protein modifications.
Microorganisms often live in iron-limiting environments due to the very low solubilities of ferric hydroxide complexes under aerobic conditions at near neutral to slightly alkaline pH values (Atkinson et al, 1998). In the cells, tissues and body fluids of animals, free iron concentration is also maintained at very low levels through the activity of iron-sequestering proteins, such as transferrin and lactoferrin, suggesting that bacterial pathogenicity depends upon an ability to acquire iron form the host. In most all aerobic and facultative anaerobic microorganisms respond to low iron conditions by producing relatively low molecular weight ferric iron-chelating siderophores to aid them in acquiring iron from their environment (Neilands, 1995). Most of these microorganisms are known to synthesize at least one siderophore and hundreds of siderophores have been structurally characterized to date (Winkelmann, 1999). In 1963, desferrioxamine B (a.k.a. Desferal, deferoxamine, desferoxamine, desferroxamine, ferrioxamine), a siderophore obtained from Streptomyces pilosus, was introduced to the clinic for removal of excess iron resulting from the supportive therapy for beta-thalassemia. A variety of oral and parenteral iron chelating agents have been devised by the clinical research community since then, though desferroxamine B remains one of the more popular treatment modalities.